PROJECT SUMMARY Lung cancer results in the largest number of cancer-related deaths in the United States. Non-small cell lung cancer (NSCLC) patient tumors overexpress NADPH oxidases that produce intracellular reactive oxygen species (ROS) throughout the cell, including the mitochondria, and play an important role in lung cancer tumorigenicity. Mitochondrial ROS have been shown to promotes lung cancer transformation. Our preliminary results demonstrate that we can transform lung cancer cells by simply overexpressing mitochondrial ROS optogenetically via the Supernova protein. Importantly, optogenetics enables precise spatiotemporal control of mitochondrial ROS without the confounding secondary effects associated with electron transport inhibitors that also alter metabolism in a ROS-independent manner. The fundamental goal of this proposal is to assess how heterogeneity in mitochondrial ROS production affects cancer-related phenotypes and signaling, and how adaptation occurs in response to selection pressure. The Aim of this proposal is to test the hypothesis that heterogeneity in mitochondrial ROS production determines whether mitochondrial ROS are tumorigenic as well as the specific downstream signaling pathways activated, and that this heterogeneity is governed by level and timing of ROS production. These investigations will parallel our linked R01 that is focused on the role of NOX enzymes and redox signaling in lung cancer by enabling us to mimic NOX activity with optogenetics, having the spatial and temporal control of ROS production that is not possible experimentally with the NOX proteins themselves. Investigating redox signaling in NSCLC may identify new regulators and therapeutic targets for intervention in lung cancer.